Despite a great deal of work, many facets of the mechanism of activated transcription remain unclear, and yet this is a central problem that must be solved for a complete understanding of gene regulation. To address this problem, we are setting up an efficient system for in vitro transcription using yeast as a model system, specifically the yeast CUP1 gene, which encodes a copper-metallothionein. We chose this gene because its biological function is relatively simple (to protect the cell against the toxic effects of copper) and its mechanism of induction is well-understood at the molecular level: copper ions bind to the DNA-binding domain of a transcriptional activator (Ace1p) which then folds and recognizes upstream activating sequences (UASs) in the CUP1 promoter. The C-terminal domain of Ace1p is a typical acidic activation domain. It is our aim to reconstitute the regulated transcription of this gene in vitro using native minichromosomes containing CUP1 isolated from induced and uninduced cells and purified yeast transcription factors. Such a system should allow us to evaluate the role of chromatin structure in the regulation of a eukaryotic gene. We have purified a small episome containing copper-induced or uninduced CUP1 in its native chromatin structure to homogeneity from yeast cells. Topological analysis and nuclease probes revealed the chromatin structure of the isolated gene. Induction resulted in the loss of a nucleosome located over the proximal upstream activating sequence (UAS) and the TATA boxes in the CUP1 promoter. The distal UAS is constitutively nucleosome-free. The presence of RNA polymerase II on induced CUP1 correlated with a major disruption of the nucleosomal organization of the transcribed region. DNase I footprinting experiments suggested the presence of a transcription complex at the promoter. Thus, a transcriptionally active gene has been isolated in its native state, providing an excellent novel model system for understanding the relationship between chromatin structure and gene regulation. These experiments provide evidence for the dynamic nature of transcriptionally active chromatin. We are now addressing questions regarding the mechanism of chromatin remodelling at the CUP1 promoter. Concurrently, we have purified many of the yeast general transcription factors. We have engineered yeast strains containing genes with six- histidine tags to facilitate purification of RNA polymerase II (holoenzyme and core enzyme), TFIIF and TFIIH. We have purified recombinant Ace1p and yTBP, yTFIIA, yTFIIB and yTFIIE. We find that RNA polymerase II core enzyme is able to recognize the CUP1 promoter without the help of other transcription factors, if the DNA is negatively supercoiled. This surprising observation reflects the fact that yeast promoters appear to be weak points in the DNA helix, which tend to melt first when subjected to superhelical stress. This has important implications for the mechanism of activation. We are now attempting to reconstitute transcription of CUP1 in minichromosomes in vitro. We are beginning a study of the histone acetylation patterns in the minichromosome. For these experiments, minichromosomes containing HIS3, a gene known to be dependent on Gcn5p (a histone acetyltransferase) for full activity, will be used. We will test the current model for the mechanism of transcriptional stimulation by Gcn5 directly using this system. - yeast CUP1 chromatin minichromosome transcription